Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal blood disorders characterized by ineffective hematopoiesis that can lead to bone marrow failure and progression to acute myeloid leukemia (AML). Aberrant DNA hypermethylation is characteristic of this disorder and, as such, DNA methyltransferase inhibitors (DMTis) are often used to treat MDS in lieu of conventional chemotherapies that have proven to be ineffective for this disease. However, even with DMTi therapy, only about one third of MDS patients achieve a clinical response leading to a survival benefit. Moreover, current DMTi-based regimens for MDS require a minimum of 6 months of treatment before a patient can be deemed resistant to this therapy. Given that only ~30-40% of patients respond to this treatment, the majority of patients will spend 6 months receiving a therapy to which they are unlikely to respond. Therefore, the identification of molecular differences that distinguish between sensitive and resistant patients at the time of diagnosis is critical in order to harness these differences for the development of molecular biomarkers predictive of DMTi response. These biomarkers would allow us to more accurately risk-stratify these patients and prevent unwarranted and ineffective therapy for resistant patients. Using next-generation sequencing technology we performed genome-wide DNA methylation, gene expression and mutational analysis on a cohort of patients with chronic myelomonocytic leukemia (CMML), an MDS/MPN overlap syndrome, including both sensitive and resistant patients to the DMTi Decitabine (DAC). We found that robust epigenetic and gene expression differences exist between DMTi responders and non-responders. From these differences, we were able to develop an epigenetic classifier that predicts response to decitabine therapy in these patients with 87% accuracy. Moreover, using RNA-seq we identified a gene expression signature associated with primary resistance to DAC in CMML. Based on these data, we hypothesize that comparable differences are likely to be found in MDS patients who are either sensitive or resistant to the DMTi Azacytidine (AZA), and that these too have the potential to be used as the basis of a molecular classifier of response as well as to identify mechanisms of primary resistance to the drug. We propose to perform genome-wide studies DNA methylation and expression studies in a cohort of 250 MDS patients treated with AZA in order to identify DNA methylation and expression biomarkers predictive of response to this agent in MDS. From these studies, we expect to be able to develop clinically useful biomarkers that can be translated to CLIA-compatible technologies that can be applied in molecular diagnostics labs.